Autophagy in chronically ischemic myocardium

Abstract

We tested the hypothesis that chronically ischemic (IS) myocardium induces autophagy, a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins, which could protect against the consequences of further ischemia. Chronically instrumented pigs were studied with repetitive myocardial ischemia produced by one, three, or six episodes of 90 min of coronary stenosis (30% reduction in baseline coronary flow followed by reperfusion every 12 h) with the non-IS region as control. In this model, wall thickening in the IS region was chronically depressed by approximately 37%. Using a nonbiased proteomic approach combining 2D gel electrophoresis with in-gel proteolysis, peptide mapping by MS, and sequence database searches for protein identification, we demonstrated increased expression of cathepsin D, a protein known to mediate autophagy. Additional autophagic proteins, cathepsin B, heat shock cognate protein Hsc73 (a key protein marker for chaperone-mediated autophagy), beclin 1 (a mammalian autophagy gene), and the processed form of microtubule-associated protein 1 light chain 3 (a marker for autophagosomes), were also increased. These changes, not evident after one episode, began to appear after two or three episodes and were most marked after six episodes of ischemia, when EM demonstrated autophagic vacuoles in chronically IS myocytes. Conversely, apoptosis, which was most marked after three episodes, decreased strikingly after six episodes, when autophagy had increased. Immunohistochemistry staining for cathepsin B was more intense in areas where apoptosis was absent. Thus, autophagy, triggered by ischemia, could be a homeostatic mechanism, by which apoptosis is inhibited and the deleterious effects of chronic ischemia are limited.

Fig. 1.

Alteration of cathepsin D in chronically IS myocardium. (Upper)2Dgel map of protein extracts from chronically IS region after six episodes. (Lower) A magnified gel region from the NI region (Left) and the IS after either three (Center) or six (Right) episodes of CS. Arrows indicate the spots after six episodes that were all later identified as cathepsin D. MW, molecular weight. Each lower image is a 6-fold magnification from the original gel.

Fig. 2.

Electron micrographs of different types of AVs observed in the six-episode chronically IS region (A–C) and six-episode NI region (D). (A) AVs containing remnants of mitochondria are demonstrated. (B) Double-membrane AVs containing recognizable cytoplasmic contents are displayed. (C) AVs containing multivesicular bodies surrounded by a sequestering membrane are demonstrated. (D) These AVs were not observed in the six-episode NI. Arrows indicate AVs. (Magnifications: ×1,400–2,000, A–D; ×5,000, A and B Insets.)

Fig. 3.

Comparison of expression level of cathepsin B and D in chronically IS myocardium at different episodes. (A and B) Western blot analysis of cathepsin D (A) and cathepsin B (B) at six episodes chronically IS vs. NI regions (Upper) and three episodes (Lower). The expression of cathepsins B and D significantly increased at six episodes (**, P < 0.01; *, P < 0.05 vs. NI). (C) The direct comparison of levels of cathepsin B (CB) and cathepsin D (CD) in chronically IS in one, three, and six episodes, again showing marked increased expression of both proteins after six episodes. (D) The elevated expression of cathepsins B and D was confirmed to occur in myocytes from the chronically IS myocardium. ADU, arbitrary densitometric units.

Fig. 4.

Enzyme activity assays of cathepsin B (A), cathepsin D (B), and β-hexosaminidase (C). Activity of both cathepsin B and D and β-hexosaminidase was much higher at six episodes in chronically IS region vs. NI region compared with three episodes vs. NI. **, P < 0.01 vs. NI; *, P < 0.05 vs NI.

Fig. 5.

Western blot analysis of Hsc73 at six episodes chronically IS vs. NI myocardium (A) and at three episodes in isolated myocytes (B) and comparing effects in necrotic tissue after myocardial infarction (IF) vs. NI regions (C). The expression of Hsc73 increased significantly in the chronically IS of six episodes (**, P < 0.01 vs. NI), but not in the IS after three episodes compared with NI. The elevation of Hsc73 after six episodes was confirmed in isolated myocytes. In contrast, the expression of Hsc73 decreased in the infarcted region. ADU, arbitrary densitometric units.

Fig. 6.

Western blot analysis of beclin 1 at three and six episodes (chronically IS vs. NI regions) (A) and in isolated myocytes from the chronically IS (B) and comparing effects in necrotic tissue after myocardial infarction (IF) vs. NI regions (C). The expression of beclin 1 was increased significantly in the chronically IS after six episodes (*, P < 0.05 vs. NI), but not after three episodes. The elevation of beclin 1 after six episodes was confirmed in isolated myocytes. In contrast, the expression of beclin 1 decreased in the infarcted region. ADU, arbitrary densitometric units.

Fig. 7.

Western blot analysis of LC3 at six episodes chronically IS vs. NI myocardium. The LC3-II/LC3-I ratio was significantly increased in the chronically IS (**, P < 0.01 vs. NI). Western blotting of actin showed equal loading of the samples.

Fig. 8.

Localization of cathepsin B and dual labeling of cathepsin B and apoptosis. (A) Fluorescent immunostaining for detection of cathepsin B in myocytes after six episodes. Cathepsin B (green) was localized to myocytes. (B and C) Dual labeling of apoptosis (TUNEL) and cathepsin B. (B) Staining for cathepsin B (blue) was more intense in areas where apoptosis was absent. (C) Conversely, myocytes associated with increased apoptosis (green) exhibited much less cathepsin B (indicated by blue and the arrow) immunostaining. (Magnification: ×430, A; ×275, B and C.)

Fig. 9.

Western blots of cathepsins B and D, Hsc73, and beclin 1 in two pigs with recovery compared after six episodes. The level of all altered proteins returned to normal in the previously IS region compared with the NI region.